Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Comparative mitochondrial genome analysis of the tobacco endophytic fungi
Leptosphaerulina chartarum and Curvularia trifolii and the phylogenetic implications
Xiao-Long Yuana,#, Min Caob,#, Guo-Ming Shena, Huai-Bao Zhanga, Yong-Mei Dua,
Zhong-Feng Zhanga, Qian Lic, Jia-Ming Gaod, Lin Xuee, Peng Zhanga,*
a Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao,
Shandong Province, China; [email protected] (X.-L.Y.); [email protected](G.-
M. S.); [email protected] (H.-B.Z); [email protected] (Y.-M.D.);
[email protected] (Z.-F.Z.)b Marine Science and Engineering College,
Qingdao Agricultural University, Qingdao, Shandong Province, China;
[email protected](M.C.)
c Nanyang Tobacco Group Co., Ltd, Nanyang , Henan Province, China;
[email protected] (Q.L.)
d Hubei Provincial Tobacco Company of China National Tobacco Corporation,
Wuhan, Hubei Province, China; [email protected] (J.-M.G.)
e WannanTobacco Group Co., Ltd, Xuancheng, Anhui Province, China
[email protected] (L.X.)
#These two authors contributed equally to this study.
Correspondence: ; [email protected] (P.Z)
© 2019 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Abstract: In tobacco plants, symbiont endophytic fungi are widely distributed in all
tissues where they play important roles. It is therefore important to determine the
species distribution and characteristics of endophytic fungi in tobacco. Here, two
parasitic fungi Leptosphaerulina chartarum and Curvularia trifolii were isolated and
identified from normal tobacco tissue. We sequenced the mitogenomes of these two
species and analysed their features, gene content, and evolutionary histories. The L.
chartarum and C. trifolii mitochondrial genomes were 68,926 bp and 59,100 bp long
circular molecules with average GC contents of 28.60% and 29.31%, respectively. The
L. chartarum mitogenome contained 36 protein coding genes, 26 tRNA genes, and 2
rRNA genes (rrnL and rrnS), which were located on both strands. The C. trifolii
mitogenome contained 26 protein coding genes, 29 tRNA genes, and 2 rRNA genes
(rrnL and rrnS). The L. chartarum 26 tRNAs ranged from 70 bp to 84 bp in length,
whereas the 29 tRNAs in C. trifolii ranged from 71 bp to 85 bp. L. chartarum and C.
trifolii mtDNAs had an identical mitochondrial gene order and orientation and were
phylogenetically identified as sisters. These data therefore provide an understanding of
the gene content and evolutionary history of species within Pleosporales.
Key words: Endophytic fungi, Leptosphaerulina chartarum; Curvularia trifolii;
mitogenomes; gene content; phylogenetic implications. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
1. Introduction
Studies examining fungal endophytes in tobacco have shown that they are widely
distributed in almost all tissues and due to their various biological functions they play
very important roles in tobacco biology [1-3]. It is therefore important to determine the
distribution of endophytic fungi species and their characteristics in tobacco.
Pleosporales, the largest order in the fungal class Dothideomycetes, includes a large
number of species, including saprobes, parasites, epiphytes, and endophytes [4].
Numerous reports have shown that many species in Pleosporales are pathogenic fungi
that can cause severe disease that have an economically and culturally adverse effect
on the global breeding industry and animal husbandry. Previous studies have shown
that many endophytic fungi that belong to the Pleosporales order can be isolated from
the healthy tissues of tobacco (Nicotiana tabacum) [5]. The family Pleosporaceae
includes a variety of species that are plant pathogens, animal parasites, and saprotrophs.
Two particularly important parasitic species are Leptosphaerulina chartarum, the
causative agent of leaf spot which has negative effects on yield throughout the world,
and Curvularia trifolii, which causes leaf blight and leaf spot on different type of feed
crops [6]. Using healthy tobacco tissues as the source, we isolated and identified these
two important parasitic species.
L. chartarum has a broad host range, usually confining its infections to Triticum
aestivum, Miscanthus giganteus, Withania somnifera, Bromus inermis Leyss, and
Panicum virgatum cv. Alamo, etc. [7-10]. It is noteworthy that the fruiting bodies of
this fungus have been reported to cause facial eczema in some animals (i.e., sheep, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
cattle, goats, and deer) due to the liver damage caused by a mycotoxin (sporidesmin)
produced by the fungus [11-13]. Several studies have also described the presence of L.
chartarum conidia in indoor air environments where asthma patients reside [14-15]. C.
trifolii, as a high-temperature pathogen that is distributed worldwide, causes leaf spot
on Trifolium alexandrinum (Berseem Clover) and leaf blight on Creeping Bentgrass
[16-18]. Moreover, C. trifolii can produce different secondary metabolites during its
culture. Among these, some show excellent anti-inflammatory and antitumor activity
[19-22]. Given that there are great similarities in life style between L. chartarum and C.
trifolii, they seem to be closely related and likely share similar genetic content and
organization.
With the rapid development of new sequencing technologies, the mitogenomes of
fungi have improved tremendously in recent years. The hundreds of available fungal
mitogenomes in the public databases have provided an efficient resource for their
classification, genetics, and analysis of their phylogenetic relationships. However, there
are just several sequences published from the Pleosporales even in the class
Dothideomycetes to which they belong [23-25]. Herein, we sequenced the complete
mitochondrial genomes of L. chartarum and C. trifolii. Furthermore, we analysed and
characterized the evolution and structural organization of L. chartarum and C. trifolii
and compared these with other species in Pleosporales. The current results allow for an
understanding of the gene content and evolutionary history of species in Pleosporales.
2. Materials and methods Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
2.1 Materials, isolation, and culture
The endophytic fungus C. trifolii was first isolated from the healthy leaves of
Nicotiana tabacum, which was collected from Enshi, Hubei, P. R. China, in July 2016.
The fungal strain L. chartarum was isolated from the fresh stems of Nicotiana tabacum,
which was also collected from Enshi, Hubei. The fungi were identified by analysis of
their internal transcribed spacer (ITS), V1, and V4 regions of rDNA, as described in
our previous report [26], as well as by their morphologies. Both strains are currently
deposited at the Tobacco Research Institute of Chinese Academy of Agricultural
Sciences. In addition, a phylogenetic analysis of C. trifolii and L. chartarum based on
the ITS1 region was performed using MEGA 6.0 [27].
2.2 DNA extraction
After isolation and purification, the hyphae of C. trifolii and L. chartarum were
scraped from the surface of the agar, after which they were ground to a fine powder in
the presence of liquid nitrogen. Genomic DNA was then extracted using a fungal DNA
extraction kit (Omega Bio-tek, Norcross, GA, USA), following the manufacturer's
instructions. Electrophoresis on 1% agarose gels was used to assess the integrity of
DNA samples. The purity and concentration of these two samples were determined
using a Nanodrop microspectrophotometer (ND-2000, Thermo Fisher Scientific,
Waltham, MA, USA) and a Qubit 2.0. fluorometer (Life Technologies, Carlsbad, CA,
USA), respectively. Genomic DNA was stored at -20°C prior to library construction.
2.3. Sequence assembly, annotation, and analysis Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
About 2 μg of genomic DNA from each strain was first fragmented into 500 bp
fragments, followed by construction of an Illumina genome sequencing libraries using
the NEBNext Ultra II DNA Library Prep Kits (NEB, Beijing, China) according to the
manufacturer's instructions. These libraries were then sequenced on an Illumina HiSeq
2500 Platform (Illumina, San Diego, CA, USA). Briefly, raw sequence reads were first
quality trimmed and adapter sequences were removed using FastQC software (version
0.11.5) [28]. Subsequently, the SPAdes 3.9.0 software package and MITObim V1.9
were used for the de novo assembly of the mitogenomes and the filling of contig gaps
[29,30]. The complete mitogenomes of C.trifolii and L.chartarum have been deposited
to GenBank (accession number of KY792993 and KY986975, respectively).
To annotate these two complete mitogenomes, their protein-coding genes (PCGs)
were identified and annotated using NCBI Open Reading Frame Finder
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html), and further confirmed with BLASTP
searches against the mitogenomes of two closely related species (Bipolaris cookei:
MF784482.1 and Pithomyces chartarum: KY792993.1). Following this, tRNAs were
identified using tRNAscan-SE 1.21 Search Server (http://lowelab.ucsc.edu/tRNAscan-
SE/) with default settings [31]. rRNA genes were also identified using multi-sequence
alignment with the GenBank sequences of B. cookie and P. chartarum mitogenomes
using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches. The base composition
of these two mitogenomes were analysed with DNAStar Lasergene v7.1
(http://www.dnastar.com/). In addition, AT skews and GC skews were determined
using (A%-T%)/(A%+T%) and (G%-C%)/(G%+C%) respectively to characterize Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
strand asymmetries in the mitogenomes. CodonW was used to analyse codon usage
frequency for amino acids in these two genomes [32]. The graphical maps of these two
mitogenomes were constructed using OGDRAW (http://ogdraw.mpimp-
golm.mpg.de/cgi-bin/ogdraw.pl) [33].
2.4 Phylogenetic analysis
To determine the phylogenetic relationships of C. trifolii and L. chartarum, current
available complete, or near-complete, mitochondrial genomes were used for
phylogenetic analyses. The class Dothideomycetes includes the orders
Botryosphaeriales, Pleosporales, and Capnodiales. Therefore, one species,
Mycosphaerella graminicola which belongs to Capnodiales, was selected as an
outgroup when the phylogenetic tree was constructed. Construction of the phylogenetic
tree was performed using a combined dataset consisting of 12 common protein coding
genes (atp6, cob, cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, and nad6)
that are encoded in C. trifolii and L. chartarum and other related mitochondrial genomes.
These genes were initially aligned with MAFFT using default settings and then
concatenated head-to-tail to form the final datasets [34]. The nucleotide substitution
models of the final datasets were selected using jModelTest [35]. Finally, the
phylogenetic tree was constructed using RAxML version 8.1.12 [36] and MrBayes [37].
For each node of the maximum likelihood (ML) tree, the bootstrap support was
calculated using 1000 replicates. For the Bayesian tree, the initial 10% of trees were
discarded as burn-in, and four simultaneous chains were run for 10,000,000 generations. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
3. Results and Discussion
3.1 Fungal isolation and identification
The strains studied here were identified as C. trifolii and L. chartarum on the basis
of their morphologies and sequences of their ITS regions (Figure S1). The BLAST
search results for ITS1 and ITS4 showed that their sequences were most similar (99.64%
and 99.82%) to the same sequences obtained from the C.trifolii isolate FUNBIO-2
(GenBank Accession No. KC415610.1) and the C .trifolii strain AL9m5_2 18S
(GenBank Accession No. KJ188716.1), respectively. Both the ITS1 and ITS4
sequences showed high identity to L. chartarum, especially for ITS4, which reached up
to 100% identify with the L. chartarum strain KNU14-16 (GenBank Accession No.
KP055597.1). Our results from the phylogenetic tree analysis supported these
alignment results. The phylogenomic investigation of ITS1 sequence demonstrated that
strain was C. trifolii. In our analysis, the phylogenetic relationships based on ITS1 also
showed that the L. chartarum strain had high bootstrap value of 99%.
3.2 General features of the newly sequenced mitochondrial genomes
The complete sequence of the L. chartarum mitochondrial genome was mapped
to be a 68,926 bp long circular molecule with an average GC content of 28.60%. The
C. trifolii mitogenome was also a typical circular DNA molecule 59,100 bp in length
with an average GC content of 29.31% (Figure 1). In the L. chartarum mitogenome, we Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
identified 36 protein coding genes, 26 tRNA genes, and 2 rRNA genes (rrnL and rrnS),
which were located on both strands. Among the protein coding genes, 11 of these
having identified functions while the remaining genes are ORFs. In total, we found 12
introns which were located in cox1&2 (7), nad5 (2), cob (2), and cox3 (1) (Table 1). In
the C. trifolii mitogenome, there were 26 protein coding genes, 30 tRNA genes, and 2
rRNA genes (rrnL and rrnS) (Table 2). We also found 22 introns which were distributed
in the atp6 (2), nad1 (4), cob (4), cox2 (4), and cox1 (4) genes.
Figure 1. Circular maps of the complete mitochondrial genomes from
Leptosphaerulina chartarum and Curvularia trifolii
Table 1. Organization of the mitochondrial genome of Leptosphaerulina chartarum
Gene R/F position Length Codon Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Intergenic From To nt aa Start Stop nucleotides
atp6 R 836 1606 771 257 AAT GTA 194
nad4 R 1849 3504 1656 552 AAT GTA 242
orf297 R 5240 6133 894 298 GAT GTA 1735
orf270 F 6871 7683 813 271 ATG TAG 737
orf393 R 8667 9848 1182 394 AAT GTA 983
cox1&2 R 9752 20506 1266 422 GAT GTA -97
orf374 R 11196 12320 1125 375 AAT AAC -9311
orf297 R 12640 13533 894 298 GAT ACA 319
orf352 R 13880 14938 1059 353 GAT AGT 346
orf363 R 15081 16172 1092 364 AAT AAC 142
orf324 R 16344 17318 975 325 AAT GCA 171
orf349 R 17553 18602 1050 350 AAT AAA 234
orf328 R 19307 20293 987 329 AAT AAG 704
trnC(gca) R 20817 20887 71 TCT GGG 523
orf156 F 22265 22735 471 157 ATG TAG 1377
orf109 R 22708 23037 330 110 GAT GTA -28
orf329 F 23702 24691 990 330 ATG TAG 664
trnR(tct) F 25731 25801 71 TTC AAT 1039
rns F 25961 27638 1678 559 TTA ATT 159
trnY(gta) F 28148 28232 85 AGG CTA 509
trnN(gtt) F 28767 28837 71 GCC GCT 534
nad6 F 29284 29958 675 225 ATG TAA 446
trnV(tac) F 29909 29981 73 AAG TTA -50
trnK(ttt) F 30009 30080 72 GAG TTG 27 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
trnG(tcc) F 30090 30160 71 ACG GTA 9
trnD(gtc) F 30163 30235 73 GGG TCG 2
trnS(gct) F 30586 30665 80 GGA CCG 350
trnW(tca) F 30995 31066 72 AAG TTG 329
trnI(gat) F 31284 31355 72 GGT TCA 217
trnR(acg) F 31359 31430 72 AGT CTT 3
trnS(tga) F 31940 32024 85 AGA CTG 509
trnP(tgg) F 32240 32312 73 CGG CGA 215
rnl F 32353 36047 3695 1231 TAT CTG 40
trnT(tgt) F 36169 36239 71 GCT GCT 121
trnM(cat) F 36261 36331 71 TGC CAT 21
trnM(cat) F 36349 36421 73 AAG TTA 17
trnL(taa) F 36916 36998 83 ATC ATA 494
trnE(ttc) F 37317 37389 73 GGC CTT 318
trnA(tgc) F 37414 37485 72 GGG CCA 24
trnF(gaa) F 37868 37940 73 GCC GCA 382
trnL(tag) F 39268 39350 83 ATG ATA 1327
trnQ(ttg) F 39645 39716 72 TAT TAG 294
trnH(gtg) F 39731 39804 74 GGT TCC 14
trnM(cat) F 40128 40198 71 GCC GCT 323
orf413 F 40936 42177 1242 414 ATG TAA 737
nad2 F 43265 45013 1749 583 ATG TAA 1087
nad3 F 45014 45436 423 141 ATG TAG 0
orf224 F 45621 46295 675 225 ATG TAA 184
orf152 F 47163 47621 459 153 ATG TAG 867
orf240 F 47835 48557 723 241 ATG TAA 213 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
nad1 F 48948 49940 993 331 ATG TAA 390
orf143 R 52108 52539 432 144 GAT GTA 2167
nad5 R 52687 58415 1272 1909 GAT GTA 147
orf302 R 54083 54991 909 303 AAT GTT -4333
orf327 R 55431 56414 984 328 AAT AAA 439
orf103 R 57780 58091 312 104 AAT GGC 1365
nad4L R 58415 58684 270 90 AAT GTA 323
orf322 R 59200 60168 969 323 AAT GTA 515
trnR(cct) R 60278 60348 71 GGA CTC 109
cob R 60443 64134 677 1230 AAT GTA 94
orf290 R 61449 62321 873 291 GAT ACA -2686
orf309 R 62812 63741 930 310 AAT GTT 490
orf109 F 64266 64595 330 110 ATG TAA 524
rnpB R 64844 65014 171 57 TAT GAC 248
orf331 R 65157 66152 996 332 GAT GTA 142
cox3 R 67443 68731 179 429 AAT GTA 1290
Table 2. Organization of the mitochondrial genome of Curvularia trifolii
position Length Codon Intergenic Gene R/F From To nt aa Start Stop nucleotides
trnR(acg) F 59 130 72 AGT CTT 117
trnS(tga) F 248 332 85 AGA CTG 230
trnP(tgg) F 563 635 73 CAG TGA 99
rnl F 735 4181 3447 1149 TAT TTA 99 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
trnT(tgt) F 4281 4351 71 GCC GCT 323
trnM(cat) F 4675 4745 71 TGC CAT 5
trnM(cat) F 4751 4823 73 AAG TTA 752
trnE(ttc) F 5576 5648 73 GGC CTT 32
trnA(tgc) F 5681 5752 72 GGG CCA 516
trnF(gaa) F 6269 6341 73 GCC GCA 378
trnL(tag) F 6720 6802 83 ATG ATA 77
trnQ(ttg) F 6880 6951 72 TAT TAA 4
trnH(gtg) F 6956 7029 74 GGT TCC 196
trnM(cat) F 7226 7297 72 GCC GCT 788
trnL(tag) F 8086 8158 73 CGG TGA 311
atp6 F 8470 12281 341 113 ATG TAG -3470
orf333 F 8812 9813 1002 334 AAA TAA 1349
orf259 F 11163 11942 780 260 ATG TAG 496
trnC(gca) F 12439 12510 72 GGG CCT 246
nad2 F 12757 14487 1731 577 ATG TAA 0
nad3 F 14488 14925 438 146 ATG TAA 1536
cox3 F 16462 17271 810 270 ATG TAA 463
orf490 F 17735 19207 1473 491 ATG TAA 1323
nad1 R 20531 24611 480 160 AAT GTA -1554
orf469 R 23058 24467 1410 470 AAT TGT 699
nad5 R 25167 27509 2343 781 AAT GTA 402
nad4L R 27912 29189 1278 426 GAT GTA 826
nad4 R 30016 31485 1470 490 AAT GTT 204
trnM(cat) R 31690 31760 71 TAA CTT 576
cob R 32337 36050 668 222 GAT GTA -2702 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
orf296 R 33349 34239 891 297 AAT AAA 486
orf257 R 34726 35499 774 258 GAT GTA 895
trnN(gtt) R 36395 36465 71 TCG CCG 508
orf996 R 36974 39964 2991 997 AAT GTA 270
trnN(gtt) R 40235 40305 71 TCG CCG 260
trnN(gtt) R 40566 40636 71 TCG CCG 395
cox2 R 41032 42968 522 174 GAT TAG -1170
orf324 R 41799 42773 975 325 AAT TTA 195
cox1 R 42969 49330 391 130 TGT GTA -5770
orf274 R 43561 44385 825 275 AAT GGA 406
orf318 R 44792 45748 957 319 AAT AAA 405
orf350 R 46154 47206 1053 351 AAT AAA 333
orf431 R 47540 49935 1296 432 AAT ATG -27
orf198 R 49909 50505 597 199 AAT GTA 1701
orf182 F 52207 52755 549 183 ATG TAG 570
trnR(tct) F 53326 53396 71 TTC AAT 64
trnR(cct) F 53461 53531 71 TTC AAA 95
rns F 53627 55255 1629 543 TTT ATT 445
trnL(taa) F 55701 55783 83 ATC ATA 16
trnY(gta) F 55800 55884 85 AGG CTA 81
trnN(gtt) F 55966 56036 71 GTT GCT 304
nad6 F 56341 56916 576 192 ATG TAA 81
trnV(tac) F 56998 57070 73 AAG TTA 32
trnK(ttt) F 57103 57174 72 GAG TTA 690
trnG(tcc) F 57865 57936 72 GCG GTA 2
trnD(gtc) F 57939 58010 72 GGG TCG 317 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
trnS(gct) F 58328 58407 80 GGA CCG 364
trnW(tca) F 58772 58843 72 AAG TTG -58844
Both the GC content and the gene content of the complete mitogenomes of L.
chartarum and C. trifolii are comparable to previously reported mitogenomes of species
in Pleosporales [25]. The genome sizes of these mitogenomes are larger than the closely
related species Shiraia bambusicola (39,030 bp), Phaeosphaeria nodorunm (49,761bp)
and Bipolaris maydis (37,250 bp), while being much smaller that the mitogenomes of
Leptosphaeria maculans (154,863bp) and Pyrenophora tritici-repentis (15,7011bp). At
least one group I and few group II introns are found in most fungal mitogenomes. For
example, there are 39 introns in the Pezizomycotina subphylum mitogenome and 2 in
the Fusarium oxysporum mitogenome [38]. However, in a previous study we did not
find any introns in the Mycosphaerella graminicola mitogenome [23]. It is noteworthy
that the differences in the number of ORFs and the presence of introns may be attributed
to the various sizes of the fungi mitogenomes, which is consistent with previous studies
[39-41].
3.3 Gene content and structural comparison
In the L. chartarum and C. trifolii mitochondrial genomes, the whole genomes
contained 36 and 26 protein coding genes, respectively. The entire length of the protein
coding genes of L. chartarum occupied 38.62% of the whole genome, while that of C.
trifolii occupied 84.02%. The overall G+C content of the protein coding genes was 29.78%
in the L. chartarum mitogenome, ranging from 21.21% (ORF109) to 43.17% (ORF270) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
(Table 3). In the C. trifolii mitogenome, the G+C content of the protein coding genes was
28.12%, ranging from 22.92% (nad6) to 32.23% (cox1) (Table 4). The mitochondrial
genome codon usage in L. chartarum and C. trifolii showed a significant bias towards A
and T, as is found other fungal species [42, 43]. In the L. chartarum and C. trifolii
mitochondrial genomes, Ile, Leu, Lys, and Phe are the most frequently encoded amino
acids, and we found that UUU, AUU, AAU, and UAU are the most frequent codons in
both of these genomes. Since these frequently used codons are exclusively comprised of
A and T (U) (Figure 2), this contribute to the high A + T content seen in most fungal
mitochondrial genomes such as in Beauveria pseudobassiana and Madurella
mycetomatis [44,45]. This preferred codon usage is strongly reflected at the third position
by the high A/T versus G/C frequencies (Table S1), which is consistent with the rich A+T
content in the whole mitogenomes.
Table 3. AT-content, AT-skew and GC-skew for mitochondrial genes of Leptosphaerulina chartarum
Feature A% C% G% T% (A+T)% AT skew GC skew
Whole genome 35.99 14.19 14.43 35.39 71.38 0.01 0.01
Protein-coding genes 35.33 14.65 15.14 34.89 70.22 0.01 0.02
cox1&2 36.73 16.43 16.19 30.65 67.38 0.09 -0.01
cox3 41.34 17.32 12.29 29.05 70.39 0.17 -0.17
cob 40.92 15.36 14.62 29.10 70.01 0.17 -0.02
nad1 28.50 12.99 15.21 43.30 71.80 -0.21 0.08
nad2 34.71 12.98 12.81 39.51 74.21 -0.06 -0.01
nad4 32.86 12.77 13.24 41.13 74.00 -0.11 0.02 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
nad4L 40.70 15.28 13.41 30.62 71.32 0.14 -0.07
nad5 40.57 15.71 10.00 33.71 74.29 0.09 -0.22
nad6 38.99 14.47 14.62 31.92 70.91 0.10 0.01
atp6 40.34 12.71 14.27 32.68 73.02 0.10 0.06
orf103 27.56 20.83 13.78 37.82 65.38 -0.16 -0.20
orf109 43.33 12.12 11.21 33.33 76.67 0.13 -0.04
orf109 36.06 8.48 12.73 42.73 78.79 -0.08 0.20
orf143 34.03 15.97 13.89 36.11 70.14 -0.03 -0.07
orf152 35.08 12.42 13.94 38.56 73.64 -0.05 0.06
orf156 44.16 13.16 11.25 31.42 75.58 0.17 -0.08
orf224 34.37 15.41 16.59 33.63 68.00 0.01 0.04
orf240 41.49 8.58 15.08 34.85 76.35 0.09 0.27
orf270 30.01 20.79 22.39 26.81 56.83 0.06 0.04
orf290 37.92 12.71 12.60 36.77 74.68 0.02 0.00
orf297 24.05 23.27 19.35 33.33 57.38 -0.16 -0.09
orf297 32.89 17.79 13.31 36.02 68.90 -0.05 -0.14
orf302 34.43 13.75 12.54 39.27 73.71 -0.07 -0.05
orf309 33.44 14.62 12.90 39.03 72.47 -0.08 -0.06
orf322 36.12 17.75 16.31 29.82 65.94 0.10 -0.04
orf324 31.79 15.08 12.41 40.72 72.51 -0.12 -0.10
orf327 33.54 16.06 12.60 37.80 71.34 -0.06 -0.12
orf328 32.22 15.50 12.46 39.82 72.04 -0.11 -0.11
orf329 40.30 9.49 15.66 34.55 74.85 0.08 0.24
orf331 37.55 12.95 14.06 35.44 72.99 0.03 0.04
orf349 33.52 14.95 13.05 38.48 72.00 -0.07 -0.07
orf352 32.58 17.85 16.81 32.77 65.34 0.00 -0.03 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
orf363 34.43 15.11 11.54 38.92 73.35 -0.06 -0.13
orf374 35.02 13.51 11.38 40.09 75.11 -0.07 -0.09
orf393 31.05 18.87 14.21 35.87 66.92 -0.07 -0.14
orf413 42.35 9.58 13.61 34.46 76.81 0.10 0.17
rnpB 28.65 14.62 14.04 42.69 71.35 -0.20 -0.02
tRNAs 41.53 24.88 33.51 48.61 90.14 -0.08 0.15
rns 33.08 15.49 22.11 29.32 62.40 0.06 0.18
rnl 35.53 13.75 20.73 29.99 65.52 0.08 0.20
NCR 37.63 12.44 12.95 36.98 74.61 0.01 0.02
Figure 2 Mitochondrial genome codon usage in Leptosphaerulina chartarum and
Curvularia trifolii
3.4 Transfer RNA and ribosomal RNA genes. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
In total, 26 tRNAs were identified in the L. chartarum mitochondrial genome,
which ranged from 70 bp (trnC(gca), trnG(tcc), trnM(cat)) to 84 bp (trnS(tga) and
trnY(gta)) in length (Table 1). The results of the secondary structure analysis showed
that all tRNAs harboured a typical cloverleaf structure, except for trnY-GTA, trnS-GCT,
trnl-TAA, trnS-TGA, and trnL-TAG (Figure 3). In addition, we found some tRNAs,
such as trnL-TAA and tRNAA-TAG, have the same anticodon. Moreover, we found
that there are three copies of tRNAM which are located in different regions of the
mitogenome. The C. trifolii mitochondrial genome contained 2,143 bp which encoded
tRNA genes, with an A + T content of 90.14%. The individual tRNAs ranged in length
from 71 bp (such as trnM(cat), trnM(cat) and trnN(gtt) etc.) to 85 bp (trnY(gta)).
Twenty-nine tRNAs in the C. trifolii mitochondrial genome carried codons for 19
amino acids (Figure 4). Among these, we found some of them existed as multiple
tRNAs (Table 2). For example, 4 tRNA genes were found to encode methionine. There
were 2, 2, and 3 different tRNA genes for leucine (trnL-TAA and trnL-TAG), serine
(trnS-GCT and trnS-TGA), and lysine (trnL-ACG, trnL-CCT and trnL-TCT),
respectively in the C. trifolii mitochondrial genome. Two ribosomal RNA genes were
found in the L. chartarum mitochondrial genome, namely rrnL and rrnS, which were
3,694 bp (with an A + T content of 65.52%) and 1,677 bp (with an A + T content of
62.40%) in length, respectively (Table 3). In the C. trifolii mitochondrial genome, the
rrnL gene was 3,447 bp long, with an A + T content of 62.37% and the rrnS gene
was1,629 bp long, with an A + T content of 62.40% (Table 4). All the tRNAs and Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
rRNAs from these two newly sequenced mitochondrial genomes are comparable to
those in related species [46,47].
Figure 3. The predicted tRNA structures of Leptosphaerulina chartarum Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Figure 4. The predicted tRNA structures of Curvularia trifolii
Table 4. AT-content, AT-skew and GC-skew for mitochondrial genes of Curvularia trifolii
Feature A% C% G% T% (A+T)% AT skew GC skew
Whole genome 35.15 14.55 14.75 35.54 70.69 -0.01 0.01
Protein-coding genes 35.86 14.59 13.53 36.02 71.88 0.00 -0.04
cox1 38.36 14.58 17.65 29.41 67.77 0.13 0.10
cox2 36.40 16.48 15.71 31.42 67.82 0.07 -0.02
cox3 29.26 15.43 16.79 38.52 67.78 -0.14 0.04
cob 39.37 15.27 16.02 29.34 68.71 0.15 0.02
nad1 41.88 15.00 13.13 30.00 71.88 0.17 -0.07
nad2 33.28 14.38 13.29 39.05 72.33 -0.08 -0.04 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
nad3 33.11 12.33 14.16 40.41 73.52 -0.10 0.07
nad4 42.31 13.54 13.67 30.48 72.79 0.16 0.01
nad4L 35.37 15.18 12.52 36.93 72.30 -0.02 -0.10
nad5 37.99 17.63 13.53 30.86 68.84 0.10 -0.13
nad6 34.72 9.72 13.19 42.36 77.08 -0.10 0.15
atp6 38.42 15.84 10.56 35.19 73.61 0.04 -0.20
orf182 35.34 12.02 15.48 37.16 72.50 -0.03 0.13
orf198 28.48 16.92 15.24 39.36 67.84 -0.16 -0.05
orf257 31.52 16.67 10.21 41.60 73.13 -0.14 -0.24
orf259 37.56 13.33 17.56 31.54 69.10 0.09 0.14
orf274 31.15 16.85 11.27 40.73 71.88 -0.13 -0.20
orf296 35.47 14.03 13.47 37.04 72.50 -0.02 -0.02
orf318 33.44 15.26 12.75 38.56 72.00 -0.07 -0.09
orf324 30.26 16.82 14.36 38.56 68.82 -0.12 -0.08
orf333 40.92 12.28 14.07 32.73 73.65 0.11 0.07
orf350 32.67 14.53 13.68 39.13 71.79 -0.09 -0.03
orf431 32.48 15.51 13.66 38.35 70.83 -0.08 -0.06
orf469 36.17 14.04 10.57 39.22 75.39 -0.04 -0.14
orf490 43.31 10.86 12.90 32.93 76.24 0.14 0.09
orf996 36.04 13.81 13.24 36.91 72.95 -0.01 -0.02
tRNAs 29.02 16.92 21.34 32.72 61.73 -0.06 0.12
rns 35.28 14.22 20.37 30.14 65.42 0.08 0.18
rnl 32.11 15.04 22.59 30.26 62.37 0.03 0.20
NCR 35.08 13.38 14.06 37.48 72.56 -0.03 0.02
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
3.5 Comparison of the mitogenome components
In order to understand the variation in mitogenome components between L.
chartarum and C. trifolii and related species, a comparative analysis of the protein-
coding gene was performed. Five representative species from Pleosporales, namely L.
chartarum, C. trifolii, Phaeosphaeria nodorum, S. bambusicola, and Didymella
pinodes, were used for the mitogenome component comparison. As shown in Figure 5,
the order of the protein-coding genes showed that gene rearrangements had occurred
among the different Pleosporales species. For the mitogenomes from L. chartarum and
C. trifolii, there were three representative regions, including block 1 (rns-nad6-rnl),
block 2 (nad2-nad4) and block 3 (cox1 and cox2) (Figure 5). In block 1, the analysis
showed that rns-nad6-rnl is a conserved region in all species. A comparison the genes
in block 2 showed that L. chartarum and C. trifolii were more similar to each other than
those in other species. The gene arrangement of nad2-nad3 in block 2 showed a diverse
range of patterns and different orientations. Moreover, some specific genes were found
to cluster together, indicating a strong relationship. For example, the gene pair nad5–
nad4L was present in all ten of the mitochondrial genomes analysed. In block 3, it is
noteworthy that cox1 and cox2 showed a different pattern in S. bambusicola and that
the cox3 gene also showed little diversity in its gene order among these five species.
The data showed that rps3 only exists in L.chartarum, while it is absent from the other
four species. A comparison of L. chartarum and C. trifolii mtDNAs indicated that these
two related species had an identical mitochondrial gene order and gene orientation. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Figure 5 Mitochondrial gene orders in five species
3.6 Phylogenetic relationship
In order to gain additional evidence for the classification of Pleosporales species and
to understand the evolutionary history of the mitochondrial genome, the complete
concatenated amino acid sequences of the 12 standard mitochondrial genes (atp6,
cox1, cox2, cox3, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and cob) were used for
a phylogenetic construction by maximum parsimony (Figure 6 ). The model GTR + I Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
+ G was chosen for the combined sequences. Both ML and Bayesian analyses
produced inconsistent topologies (Figure 6). M. graminicola was located in the basal
position of the phylogenetic tree. In the Pleosporales group, L. chartarum and
C.trifolii were found to be sister groups with high confidence (100% for bootstrap and
1 for BI). This clade clustered with the clade containing the other five species P.
tritici-repentis, P. nodorunm, L. maculans, D. pinodes, and B. maydis. Among this
clade, L. maculans was located in the medium position of these species and was
therefore separated from (P. tritici-repentis, P. nodorunm) and (D. pinodes and B.
maydis). It appears that S. bambusicola is at a greater genetic distance from other
species in Pleosporales. The results from our study are therefore consistent with
previous studies [25]. As mentioned above, there are just several mitogenomes have
been published from the Pleosporales even in the class Dothideomycetes belongs to
[23-25]. The complete mitogenomes of L. chartarum and C. trifolii will provide more
effective information for us to understand the phylogenetic relationship of species
among Pleosporales. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
Figure 6 Phylogenetic trees constructed by the ML and BI methods based on 12
common protein coding genes. Species used in this study. Leptosphaerulina chartarum
and Curvularia trifolii (in this study); Shiraia bambusicola (NC_026869.1); Didymella
pinodes (NC_029396.1); Bipolaris maydis (AIDY01000067 and AIDY01000043));
Leptosphaeria maculans (FP929115); Pyrenophora tritici-repentis (NW002475730);
Phaeosphaeria nodorunm (NC009746); Mycosphaerella graminicola (NC010222).
4. Conclusions
Fungal endophytes in tobacco are widely distributed in almost all tissues and play
a very important role because they have a variety of different biological functions.
Therefore, it is important to determine the species distribution and characteristics of
endophytic fungi in tobacco. Using healthy tissues from tobacco as the source, we Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
isolated and identified two endophytic fungi namely L. chartarum and C. trifolii and
obtained the sequences of their mitogenomes. We successfully annotated their protein-
coding genes, compared their gene content and A+T content, and then constructed their
phylogenetic relationship with other available species in Pleosporales. These data
provide us with new insights to determining the species distribution and characteristics
of endophytic fungi in tobacco.
Author Contributions: P. Z and Z.-F.Z. conceived and designed the project. X.-L.Y.
and M.C. performed the experiments. G.-M.S., H.-B. Z and Y.-M.D. drafted and
revised the manuscript. Q.L, L.X., J.-M.G, and Y.-M.D. analyzed and interpreted the
data. All authors have read and approved the final manuscript.
Funding: This work was supported by the Natural Science Foundation of China (grant
number 31700295),the Science Foundation for Young Scholars of Institute of
Tobacco Research of Chinese Academy of Agricultural Sciences (grant number
2016A02),and the Agricultural Science and Technology Innovation Program (Grant
No. ASTIP-TRIC05).
Conflicts of interest:The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 July 2019 doi:10.20944/preprints201907.0119.v1
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